Lysosomes can play a major role in breast cancer invasion and metastasis, because they are a key mediator of protease routing, regulation, and secretion. Lysosomes contain diverse proteases that breast cancer cells utilize to invade through the extracellular matrix. Thus, non-invasive imaging of lysosomes may provide an innovative strategy for predicting and delineating the metastatic potential of breast tumors. We have recently developed novel methods to optically image lysosomes in living human breast cancer cells, and to quantitate specific lysosomal parameters. These methods now enable us to test our hypothesis that lysosomes, and lysosomal trafficking mediated by cytoskeletal components, plays an important role in breast cancer invasion and metastasis. Our specific aims are: (1) To determine whether breast cancer cells recruit lysosomes to their cell surface to invade through the ECM, thereby either facilitating exocytic or endocytic processes, or both. (2) To assess the influence of the tumor microenvironment, characterized by hypoxia and extracellular acidity, on lysosomal trafficking in invading breast cancer cells, thereby evaluating the role of cytoskeletal components involved in lysosomal trafficking. (3) To develop and validate in vivo optical imaging of lysosomal parameters in human breast tumor xenograft models. Non-invasive, state-of-the-art cellular and in vivo optical imaging, such as confocal, total internal reflection, and multiphoton fluorescence microscopy will be employed. A panel of human breast cancer cell lines representing different stages of invasiveness and metastatic potential will be utilized in cell studies and tumor xenograft mouse models. Our research proposal will test whether imaging lysosomes in breast cancers can predict the metastatic potential of these tumors. Disruption of lysosomal trafficking may prove useful, therapeutically, to prevent metastasic disease.